Human cytomegalovirus (HCMV) is the major viral cause of birth defects and affects the central nervous system. It is likely that gestational age an the stage of differentiation of the susceptible neural cells at the time of infection will determin the severity of the disease. A primary goal of this proposal is to determine mechanistically how HCMV infection disrupts the homeostasis of cells of the neural lineage, and impairs survival and differentiation. The focus is on glycolysis, induction of ROS, and autophagy. A second goal is to determine whether we can interfere with the viral manipulation of these pathways, and thus inhibit the viral infection and restore cell survival and appropriate differentiation. For these studies we will use human primitive neural stem cells (pNSCs) that have been derived from approved embryonic stem cells. Established protocols will be used to differentiate the pNSCs to neurons and astrocytes. Cells will be infected with HCMV at various stages during differentiation as well as after differentiation to neurons and astrocytes. In Aim 1, we will determine how oxidative stress and altered metabolism in infected cells of the neural lineage impairs survival and differentiation. Various strategies will be employed to counter the effects of reactive oxygen species (ROS) and redirect glucose metabolism through the pentose phosphate pathway to maintain an antioxidant environment. In Aim 2, we will elucidate the effects of viral infection on autophagy. We predict that analogous to the effects of the infection in fibroblasts, HCMV will inhibit autophagy and this will contribute to altered differentiation and cell death. Since the actve form of Vitamin D3 has been shown to induce autophagy and impair HIV replication, we will determine whether raising the levels of active vitamin D3 will inhibit HCMV infection and have a positive impact on the health of the cell. We expect that these studies will provide great insight into the effects of HCMV on cellular metabolism and neurogenesis and serve as a foundation for future therapeutic efforts in preventing the birth defects due to congenital HCMV. In addition, the results will provide important information regarding how disseminated HCMV infection in immunosuppressed individuals might affect the function and differentiation of transplanted allogeneic neural stem cells, a serious potential problem that has received little attention thus far.